Selenium carrier conjugates

ABSTRACT

The invention provides a method for making a selenium-carrier conjugate by covalently attaching (i) an organic selenium compound selected from the group consisting of RSeH, RSeR, RSeR&#39;, RSeSeR and RSeSeR&#39;, wherein R and R&#39; are each an aliphatic residue containing at least one reactive group selected from the group consisting of aldehyde, amino, alcoholic, carboxylic, phosphate, sulfate, halogen or phenolic reactive groups and combinations thereof, to (ii) a carrier having a constituent capable of forming a covalent bond with said reactive groups of said selenium compound to produce a selenium-carrier conjugate which is capable of specific attachment to a target site. The carrier may be a protein, such as an antibody specific to a bacteria, virus, protozoa, or cell antigen, including without limitation, cell surface antigens, a peptide, carbohydrate, lipid, vitamin, drug, lectin, plasmid, liposome, nucleic acid or a non-metallic implantable device, such as an intraocular implant or a vascular shunt. The attachment of selenium compounds of the configurations described above to carriers when presented to either endogenous thiols, such as glutathione which occurs in all aerobic living cells, or exogenous thiols, such as glutathione or cysteine, produces superoxide (O 2 . - ), hydrogen peroxide, the hydroxyl radical (.OH) and other cytotoxic reactive oxygen species so as to collectively form a localized free radical pharmacology based upon the catalytic selenium anion, RSe - .

This application is a divisional of application Ser. No. 08/432,584filed May 9, 1995 now U.S. Pat. No. 5,783,454, which is acontinuation-in-part of application Ser. No. 08/243,704, filed May 17,1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to selenium compounds, and morespecifically to selenium compounds which, when covalently attached tofunctional and site directed molecules and devices, produce superoxideand other reactive compounds in the presence of thiols.

2. Description of the Invention Background

Selenium (Se) is among the most toxic of all known minerals. Itstoxicity symptoms in horses were most likely described by Marco Polowhile traveling the silk road in China. In the 1920's, loss of livestockin parts of the western and central United States was severe. Thoselosses of livestock were investigated by the United States Department ofAgriculture Experiment Station in South Dakota. In 1934, the cause ofthe loss of livestock was traced by the Experiment Station to theelement selenium which was high in certain soils and high secondarily inplants from several species of Astragalus (vetch), Xylorrhiza (woodyaster), conopsis (goldenrod) and Stanleya (Prince's Plume). Ingestion ofthese and other Se containing plants by livestock often proved to befatal.

Throughout the period of time between the discovery of selenium toxicityin livestock in 1934 and 1988, many hypotheses were put forth to explainthe mechanism by which many but not all compounds of selenium weretoxic. None of these theories of selenium toxicity proved satisfactoryin fully explaining why selenium was toxic. In 1989, Seko, Y. E., Saito,Y. Kitahare, J. and Imura, N., "Active oxygen generation by the reactionof Selenite with reduced glutathione in vitro," In: Proceedings of thefourth international symposium on selenium and medicine (ed., Wendel,A.) pp. 70-73, Springer-Verlag, Heidelberg, Germany, (1989), reportedthat selenite, (SeO₃), an inorganic form of Se, reacted with a thiol,glutathione, (GSH), to produce superoxide (O₂.⁻). Since superoxide is aknown toxicant, this raised the possibility that all selenium compoundsthat are toxic might generate superoxide. Through the testing of manyselenium compounds, it was found that the inorganic compounds, SeO₃ andselenium dioxide (SeO₂) were able to generate O₂.⁻ and hydrogen peroxide(H₂ O₂) when presented with a thiol, such as glutathione, cysteine(CysSH), or dithiothreitol D(SH)₂. Furthermore, it was found that alldiselenides of the composition RSeSeR tested likewise would generateO₂.⁻ and H₂ O₂ when presented with any of the before mentioned thiols.

In 1947, Feigl, F. and West, P. W. "Test for selenium based on acatalytic effect", Analytical Chemistry, vol. 19, pp. 351-353 (1947),reported that selenium could catalyze a redox reaction involving sulfideoxidation. This soon became a common test for selenium using methyleneblue. This reaction was further studied by others using differentselenium compounds and thiols, demonstrating catalysis for some but notall selenium compounds. See, West, P. W. and Ramakrishna, T. V. "Acatalytic method for determining traces of selenium," AnalyticChemistry, vol. 40, pp. 966-968 (1968); Levander, O. A., Morris V. C.,and Higgs, D. J. "Selenium as a catalyst for the reduction of cytochromeC by glutathione", Biochemistry, vol. 12, pp. 4591-4595 (1973), Rhead,N. J. and Schrauzer, G. N., "The selenium catalyzed reduction ofmethylene blue by thiols", Biorganic Chemistry, vol. 3, pp. 225-242(1974). The selenium catalytic activity of selenocystine (RSeSeR) in thepresence of thiols was reported in 1958. It is now believed that all ofthe foregoing reactions of selenium compounds produce superoxide. See,Xu, M., Zhang, L., Sun, E. and Fan, H., "Studies on the interaction oftrace element selenium with oxygen free radical," Advances in FreeRadical Biology and Medicine, vol. 1, pp. 35-48 (1991); Xu, H., Feng,Z., and Yi, C., "Free radical mechanism of the toxicity of seleniumcompounds," Huzahong Longong Daxus Xuebao, vol. 19, pp. 13-19 (1991);Kitahara, J., Seko, Y. and Imura, N., "Possible involvement of activeoxygen species in selenite toxicity in isolated rat hepatocytes,"Archives of toxicology, vol. 67, pp. 497-501 (1993); Chaudiere, J.,Courtin, O. and Ledaire, J., "Glutathione oxidase activity ofselenocystamine: a mechanistic study," Archives of Biochemistry andBiophysics, vol. 296, pp. 328-336 (1992).

Selenium and a number of its compounds have been known since the early1970's to possess anti-cancer properties. It has been generallyrecognized that selenite and selenium dioxide are good anti-canceragents in vitro and in experimental animals and that the compounds arealso cytotoxic to both cancer and normal cells in vitro. U.S. Pat. No.5,104,852 to Kralick et al. describes the use of selenodiglutathione andother selenodithiols of the configuration (GSSeSG) to treat cancer.Selenodiglutathione is the product of the reaction between selenite orselenium dioxide with glutathione. The compound, selenodiglutathione,has been isolated. U.S. Pat. No. 5,104,852, however, does not describethe mechanism of action by which selenodiglutathione and like compoundsare useful in treating cancer.

In 1982, the interaction of selenite and selenocystine with glutathionein the cytotoxicity and lysis of rat erythrocyte membranes was describedby Hu, M. L. and Spallholz, J. E., "In vitro hemolysis of raterythrocytes by selenium compounds", Biochemical Pharmacology, vol. 32,pp. 857-961 (1983). This cytotoxicity, as revealed by scanning electronmicroscopy of rat erythrocytes, caused the erythrocyte membranes tobecome burred, the cells to quadruple in size and lyse similar to thatdescribed by Kellogg, E. and Fridovich, I., "Liposome oxidation anderythrocyte lysis by enzymically generated superoxide and hydrogenperoxide," J. Biol. Chem., vol. 252, pp. 6721-6728 (1977). Thistoxicity, however, was not expressed by selenomethionine, a compoundpossessing the configuration RSeR. In 1991, an article by Yan, L. andSpallholz, J. E., "Free radical generation by selenium compounds," FASEBJ., vol. 5, p.A581 (1991), showed a dose responsive toxicity of severalselenium compounds to a human mammary tumor cell line. Additionalinvestigations using lucigenin chemiluminescence and luminolchemiluminescence revealed a dose response in O₂.⁻ and H₂ O₂ generatedchemiluminescence by selenite, selenium dioxide and all seleniumcompounds tested of the configuration RSeSeR. It was found furthermore,that selenium compounds in the presence of either tumor cells orglutathione alone produced superoxide and H₂ O₂. Chemiluminescence fromthe reactions of lucigenin with O₂.⁻ or luminol with H₂ O₂ could bequenched by the native enzymes superoxide dismutase (SOD), catalase (CT)or glutathione peroxidase (GSHPx). Denatured enzymes would not quenchthese reactions, confirming the generation of the free radical (O₂.⁻)and H₂ O₂ by selenium compounds and thiols. All of this selenium freeradical chemistry has been reviewed by Spallholz, J. E., "On the natureof selenium toxicity and carcinostatic activity," Free Radical Biologyand Medicine, vol. 17, pp. 45-64, (1994).

A summation of this large body of experimental data on seleniumtoxicity, catalysis and carcinostatic activity is as follows:

1) The selenium compounds, SeO₂ and SeO₃, react with thiols to produce aselenodithiol of the configuration (RSSeSR). This compound is not toxicper se nor is it carcinostatic. The toxic carcinostatic form of RSeR isthe reduced selenide anion, RSe⁻. This selenopersulfide form of Se iscatalytic as shown by the inhibition of both catalysis and superoxidegeneration by iodoacetic acid and mercaptosuccinic acid.

2) Selenium compounds of the configuration (RSeSeR) or (RSeSeR') reactwith thiols to produce the reduced selenite anion RSe⁻ or R'Se⁻. Thisselenopersulfide form of Se is catalytic as shown by the inhibition ofboth catalysis and superoxide generation by iodoacetic acid andmercaptosuccinic acid.

3) Organic selenium catalysts by the configuration RSe⁻ theselenopersulfide anion, is catalytic in the presence of thiols and RSe⁻continues to generate superoxide (O₂.⁻) ion as long as sufficientconcentrations of O₂.⁻ and thiol are in the medium. Selenium compoundsderived from selenite or selenium dioxide reacting with glutathione(GSH) are converted to elemental selenium (Se) follows; SeO₃(SeO₂)+2GSH-2GSSeSG-2GSSG+Se. Elemental selenium (Se) is non-catalyticand not toxic.

4) Compounds of selenium of the configuration RSe⁻ are toxic due to thecatalytic acceleration of thiol oxidation which produces O₂.⁻, H₂ O₂ andthe more toxic free radical, the hydroxyl radical (.OH). This chemistryhad been discussed by Misra, H. P., "Generation of superoxide freeradical during the auto-oxidation of thiols," J. Biol. Chem., vol. 249,pp. 2151-2155 (1974) for the spontaneous oxidation of thiols. Theassociation of rapid thiol catalysis by selenium compounds of theconfiguration RSe⁻ and the toxicity from which it produced free radicalsand reactive toxic oxygen products was recognized in 1992 by one of theinventors.

The use of selenium for the treatment of experimental cancer in animalsand cancer in humans in vivo has been extensively described by manyauthors, such as Milner, J. A., Greeder, G. A., Poirier, K. A.,"Selenium and transplantable tumors," (Spallholz, J. E., Martin, J. L.,Ganther, H. E., eds.) Selenium in Biology and Medicine, AVI PublishingCo. (1981); Ip, C. and Ganther, J. E., "Relationship between thechemical form of selenium and anticarcinogenic activity," CRC Press,Inc., pp. 479-488 (1992); Caffrey, P. B. and Frenkel, G. D. "Selenitecytotoxicity in drug resistant and nonresistant human ovarian tumorcells," Cancer Research, vol. 52, pp. 4812-4816 (1992); Schrauzer, G.N., "Selenium: Mechanistic aspects of anticarcinogen action", Biol.Trace Elem. Res., vol. 33, pp. 51-62 (1992); Yan, L. and Spallholz, J.E. "Generation of reactive oxygen species from the reaction of seleniumcompounds with thiols and mammary tumor cells," BiochemicalPharmacology, vol. 45, pp. 429-437 (1993). The use of selenium as acytotoxic agent to both normal cells and cancer cells in vitro has beendescribed in U.S. Pat. No. 5,104,852 for the injection ofselenodiglutathione into a tumor mass to kill tumor cells. In U.S. Pat.No. 4,671,958, Rodwell et al. described many antibacterial drugs, 3antiviral drugs, 1 antifungal drug, 7 antineoplastic drugs, 3radiopharmaceuticals, 3 heavy metals and 2 antimycoplasmals as drugs forantibody mediated delivery. The pharmacology for all of these drugswhich are listed in Table 1 of U.S. Pat. No. 4,671,958 is generallyunderstood. Table 1 of the Rodwell et al. patent does not containselenium because its pharmacological action as a free radical generatorof (O₂.⁻) and other reactive oxygen molecules was not understood orknown at that time.

Viral infections are difficult to treat because viruses lack an uptakemechanism by which agents designed to kill or damage the virus can betaken into the virus. Even when it is known that a person has beeninfected with a virus, it has been necessary heretofore to wait for thevirus to infect cells in the host and reproduce in the cells. The cell,which does have an uptake mechanism, will takeup the agent. Viralreproduction can only then be blocked. In practice, the viral infectionhas usually spread by the time treatment is possible. This isparticularly devastating with HIV viral infections, the causative agentfor acquired immune deficiency syndrome (AIDS). Often, an infectedperson knows that he or she has been exposed to the virus but is unableto stop the spread of the virus until that person's cells have beeninfected.

Once-reliable antibiotics for treating bacterial infections are fallingby the way side. With the emergence of many conventional drug-resistantstrains of bacteria, such as those identified in the Mar. 28, 1994 issueof Newsweek magazine and set forth below in Table 1, the need for a newagent for combating infectious diseases is becoming critical.

                  TABLE 1                                                         ______________________________________                                        DRUG RESISTANT BACTERIA                                                                                     ANTIBIOTICS THAT                                  BACTERIA DISEASES CAUSED NO LONGER WORK                                     ______________________________________                                           Enterococcus                                                                                Blood poisoning,                                                                         Aminoglycosides,                                             surgical infections  cephalosporins,                                   erythromycin,                                                                 penicillin,                                                                   tetracycline,                                                                 vancomycin                                                                  Hemophilus         Meningitis, ear    Chloramphenicol,                        influenza           infections,          penicillins,                                   pneumonia,          tetracycline,                                             sinusitis            trimethorprirm/                                  sulfamethoxazola                                                            Mycobacterium       Tuberculosis      Aminoglycosides,                        tuberculosis                             ethambuto1,                            isoniazid,                                                                   pyrazinamide,                                                                  rifampin                                                                    Neisseria           Gonorrhea          Penicillins,                           gonorrhea                                 spectinomycin,                                              tetracycline                                          Plasmodium   Malaria              Chloroquine                                 falciparurn                                                                   Shigella            Severe Diarrhea      Ampicillin,                          dysenteriae                                chloramphenicol,                     tetracycline,                                                                 trimethoprim/                                                                 sulfamethoxazole                                                            Staphylococcus      Blood poisoning,    All but                               aureus            pneumonia, surgical Vancomycin                               infections                                                                   Streptococcus       Meningitis,          Aminoglycosides,                     pneumoniae         pneumonia           cephalosporins,                          chloramphenicol,                                                              crythromycin,                                                                 penicillins,                                                                  tetracycline,                                                                 trimethorprim/                                                                sulfamethoxazola                                                          ______________________________________                                    

It is an object of the invention to provide a new bacterialcidal andviralcidal agent.

It is a further object of this invention to provide a methodology to useof the aforementioned free radical technology as bacterialcidal orviralcidal agents. It is a further object of the present invention toprovide a method for directing the localized production of superoxideand descendant species thereof for selective destruction or modificationof cells, tissue, membranes or extracelluler fluids to combat a varietyof localized problems, from infections, to cancer, to post surgicalclotting and fibrosis.

SUMMARY OF THE INVENTION

The present invention comprises the organic covalent chemical attachmentof organic selenium compounds of the configuration, RSeH, RSeR,RSeR',RSeSeR or RSeSeR' to various carrier molecules, where R and R' areeach selected from the group consisting of aliphatic residues containingone or more aldehyde, carboxylic, amino, alcoholic, phosphate, sulfate,halogen or phenolic reactive groups, and combinations thereof, such as--(CH₂)_(n) HN₂, --(CH₂)_(n) COOH, --(CH₂)_(n) --.O slashed., wherein nis an integer equal to or greater than 1. R and R' can be the same ordifferent. The carrier molecules, which have a complementary reactiveconstituent capable of forming a covalent bond with the reactive groupsof the selenium compound, include monoclonal antibodies, polyclonalantibodies, polypeptides, peptides, carbohydrates, lipids, vitamins,drugs and other organic carriers or devices for the treatment ofdiseases caused by pathogens, such as viral, bacterial, protozoan,richetcial, yeast, mycoplasma, fungi or other pathogens and diseases ofeither known or unknown etiology, such as cancers and glaucoma. Thepresent invention is directed to the treatment and cure of a widespectrum of cancers, bacterial, viral and other biotic infections.

An interesting discovery of the invention is that the tissue, cell orbodily fluid provides the reducing power for the generation ofsuperoxide (O₂.⁻). However, should additional reducing power be neededin vivo it can be supplied by exogenous glutathione or cysteineaccording to known techniques.

The invention also includes a method for treating cancer tumors andpathogenic infections by means of administering an effective amount of aselenium-carrier conjugate for localized delivery and attachment to atarget site comprising cancer tumors and pathogens having endogenousthiol compounds. The selenium-carrier conjugate comprises (i) an organicselenium compound selected from the group consisting of RSeH, RSeR,RSeR',RSeSeR and RSeSeR',wherein R and R' are the same or different andeach is an aliphatic residue containing at least one reactive groupselected from the group consisting of aldehyde, amino, alcoholic,carboxylic, phosphate, sulfate, halogen or phenolic reactive groups andcombinations thereof, covalently attached to (ii) a carrier having aconstituent capable of forming a covalent bond with the reactive groupsof the selenium compound to produce the selenium-carrier conjugate. Thecarrier is capable of specific attachment to the target site for thelocalized generation of superoxide for localized destruction at thetarget site. The invention also includes a method for preventing tissuedamage associated with the use of implants and the prevention ofunwanted scarring in surgical procedures. The aforementioned organicselenium compound is covalently attached to the surface of animplantable device or a stationary matrix, resulting in aselenium-coated device or carrier, as the case may be, for the localizedgeneration of superoxide at the implant, or surgical site to inhibit theformation of scar tissue or other undesirable cellular growth.

The invention also comprises the attachment of these same seleniumadducts for immunoassays and other quantitative analytical work asdescribed by U.S. Pat. No. 4,341,757, dated Jul. 23, 1982 which isincorporated herein by reference. The invention includes, for example, amethod for detecting the presence of a compound of biological interestcomprising the steps of incubating together a mixture of a samplesuspected of containing a compound of biological interest and aselenium-carrier conjugate as described above, wherein the carrier iscapable of specific attachment to the compound of biological interest,to bind the selenium-carrier conjugate to a determinant on the compoundof biological interest. Then, any unbound selenium-carrier conjugate isremoved. Thereafter, the method includes the steps of adding a thiolcompound to the mixture to react with the selenium to generatesuperoxide, then adding a reporting agent to the mixture. The reportingagent is capable of reacting with superoxide. Finally, the reactionbetween the reporting agent and superoxide is detected. The reportingagent may be, for example, lucigenin, methylene blue or cytochrome C.Compounds of biological interest may be proteins, vitamins, hormones,enzymes, antibodies, polysaccharides, cell and tissue antigens, thepathogens described herein, drugs or other blood cell or intra orextracellular fluids. Antibodies specific to any of those compounds canbe made by well known techniques. Such antibodies can be used as thecarrier for attachment to the selenium compound of the invention.

The attachment of selenium compounds of the configurations describedabove to carriers, diagnostics or devices when presented to eitherendogenous thiols, such as glutathione which occurs in all aerobicliving cells, or exogenous thiols, such as glutathione or cysteine,produces superoxide (O₂.⁻), hydrogen peroxide, the hydroxyl radical(.OH) and other cytotoxic reactive oxygen species so as to collectivelyform a localized free radical pharmacology and new pharmacopeia basedupon the catalytic selenium anion, RSe⁻ Because superoxides are sodeadly to cells, the body has natural mechanisms to destroy thesuperoxides, i.e., with dismutase. Thus, the superoxide radical, O₂.⁻,has a relatively short half life and will degrade. H₂ O₂ and OH aresecondarily produced and are slightly longer lived. As used herein, forbrevity, superoxide will include O₂.⁻ and its descendent oxygen species.Because of the short life, O₂.⁻ must be generated at or near the site ofintended destruction. The covalent attachment of selenium compoundswhich produce the RSe⁻ anion provide for a new analytical chemistrybased upon the generation and detection of superoxide (O₂.⁻) usingchemiluminescence or the reduction of various dyes, such as methyleneblue or cytochrome C. Methylene blue and cytochrome C in the oxidizedform may be reduced by selenium attached to a receptor molecule, throughthe generation of superoxide. The amount of reduced methylene blue orcytochrome C can be measured spectrophotometrically and quantitated,thereby reflecting the concentration of the molecule to which seleniumis attached.

This invention provides preferably for a small molecular adduct, RSeH,RSeR, RSeR', RSeSeR or RSeSeR', the detector group for analyticalchemistries using chemiluminescence or chemical dyes by producingsuperoxide (O₂.⁻); and the generator of superoxide (O₂.⁻) whereby it isonly toxic in a very localized area produced by the conjugate RSe⁻ andwhich is determined by the binding of a carrier molecule or localized bya stable insoluble matrix. The present invention substantially overcomesthe generalized problem of systemic selenium toxicity in the treatmentof diseases such as metastatic cancer and demonstrates a new appliedfree radical pharmacology. This invention represents a major advancementin the application of selenium toxicity in the treatment of diseases andin the extension or analytical sensitivity and ease of detection forstable isotopic immunoassays. The targeting superoxide free radicalpharmacology, based upon the addition of selenium to targeting moleculesof the present invention holds promise for a new bacterial pharmacopeia.The same is also true for viral infections whereby conjugation of theappropriate selenium configuration to a viral targeting agent, such anantibody or binding peptide will render a virulent virus non-virulent.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood by reference to the figures inwhich:

FIG. 1 is a graph demonstrating the percent hemolysis of humanerythrocytes produced by Ab-Se conjugates, native Ab, and combinationsof the two;

FIG. 2 is a graph showing the cytotoxicity of a covalentantibody-selenium conjugate;

FIG. 3 is a bar graph showing cell growth, as measured by tritiatedthymidine incorporation, on plastic treated with CO₂ plasma with andwithout subsequent covalent selenocystamine coupling;

FIG. 4 is a bar graph showing the growth of cells on plastic treatedwith CO₂ plasma with and without subsequent covalent coupling ofselenocystamine for varying treatment times; and

FIG. 5 is a bar graph showing cell growth in the presence and absence ofcovalently attached selenium treated cellulose sponges as measured bytritiated thymidine incorporation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the covalent attachment of selenium adducts ofthe configuration RSeH, RSeR, RSeR', RSeSeR or RSeSeR' to carriermolecules, diagnostics and devices. The Se-carrier conjugate thusproduced is administered by injection or ingestion and carried by normalphysiologic means to a target site or target molecule, or surgicallyimplanted at a target site, whereupon superoxide (O₂.⁻) is generatedwhen the Se-carrier conjugate reacts with endogenous thiols on thesurface of the targeted local tissue, bacteria, virus, protozoa or othertargeted compounds. The selenium toxicity produced by the conjugate RSe⁻is very localized because it is determined by the specific binding ofthe carrier molecule or localized by the stable insoluble matrix of animplant. R and R' are each selected from the group consisting ofaliphatic residues containing one or more aldehyde, carboxylic, amino,alcoholic, phosphate, sulfate, halogen or phenolic reactive groups, andcombinations thereof, such as --(CH₂)_(n) HN₂, --(CH₂)_(n) COOH,--(CH₂)_(n) --.O slashed., wherein n is an integer greater than 1, andpreferably between about 1 and 50, and more preferably between about 3to 5. R and R' can be the same or different. The R groups themselveshave no real role in the method of the invention, other than to providereactive groups to bind to the carrier and to protect the selenium untilit reaches the target sites. Accordingly, the length of the aliphaticchain is not important. The preferred molecule weight of the compound isabout 1000 or less, but higher MWs will be suitable. Representativeexamples of selenium compounds include, but are not limited to NH₂ CH₂CH₂ SeCH₃ (RSeR'), NH₂ CH₂ CH₂ SeCH₂ CH₂ NH₂ (RSeR), NH₂ CH₂ CH₂ SeSeCH₂CH₂ NH₂ (RSeSeR), NH₂ CH₂ CH₂ SeSeCH₂ CH₂ NH-cellulose (RSeSeR') andselenocystamine. The RSeH, RSeSeR, and RSeSeR' configurations arepreferred. These selenium compounds are covalently or otherwise attachedto any polyclonal or monoclonal antibody which, when brought intocontact with thiol, can then generate superoxide (O₂.⁻), H₂ O₂, orhydroxyl radical (.OH) or any other reactive oxygen specie. The thiolscan be exogenous thiols added for example, to a competitive immunoassay,endogenous thiols found in membranes, cellular cytoplasm orextracellular fluids. If native thiols are insufficient, exogenouslysupplied glutathione, glutathione derivatives, cysteine or other thiolcan be used expressly for the generation of superoxide. Theselenium-carrier conjugate is used to treat, in a pharmacologicalmanner, cancer, both primary and metastatic, infections and diseasescaused by all viruses of all plant, animal or human origin, all bacteriaof all plant, animal or human origin, all protozoans of all plant,animal or human origin and other pathogens. The antibody-seleniumconjugate, for example, will specifically bind to the virus, bacteria,protozoa or cancer cells and catalyze the production of superoxide, H₂O₂ and other reactive oxygen species. Viruses have surface proteins towhich the antibody selenium-conjugate can attach. The selenium reactswith thiols in those surface proteins to generate the superoxide on thesurface of the virus. The lack of an uptake mechanism in the virus isnot important because the damage is done at the viral surface.

When the organic Se compound is attached to a site directed antibodymolecule, it is believed to typically bind at the F(Ab⁻)2 fragment ofthe antibody. Other fragments of the antibody, natural or synthetic, canbe the site of attachment, such as carbohydrates or modified aminoacids.

The site directing molecule that carries selenium can be any protein,polypeptide, peptide, carbohydrate, lipid, drug, vitamin, hormone,lectin, plasmid, liposome, nucleic acids, or other site directed organicmolecules for which their exists a receptor. Such site directedsubstances are known to those skilled in the art. Whatever the locationof the problem, a constituent that will be directed to the site ischosen as the carrier. The selenium is introduced into an antibody orother site directed molecules by chemical modification of existingmoieties such as the amino acid serine, to generate the covalentconfigurations, RSeH, RSeSeR, RSeSeR' or RSe⁻.

The selenium may be covalently attached to any protein, antibody, haptenor matrix to be used in any immunoassay or competitive binding assaywhereby superoxide (O₂.⁻) or its descendant reactive oxygen species areused as a detection system using chemiluminescence or chemical dyes ormolecules which react with O₂.⁻ or H₂ O₂ as the reporter of the unknownselenium adduct concentrations.

The selenium compounds may be attached to any solid or stationary matrixsuch as a cellulose pad, protein pad, other carbohydrate pad, plastic orother polymer matrix or a biocompatable fibrous matrix for the purposeof generating superoxide (O₂.⁻) and its descendent reactive oxygenspecies when the matrix is implanted. The device should not be metallic,but may be an organometallic compound or a metal coated with an organocompound to which the selenium compound can attach. The seleniumattached to the insoluble matrix inhibits cell growth in the localizedarea of the matrix due to the localized generation of superoxide.

Diagnostics, as used herein, refers to molecules to which selenium hasbeen attached, which molecules are then specifically bound to anothermolecule for use in diagnostic testing for the generation andmeasurement of superoxide radicals. Examples of diagnostic tests withwhich the Se-carrier conjugates of the present invention areparticularly well suited include competitive binding assays, directbinding assays, immunoassays, and histologic assays, such as nitrobluetetrazolium reduction from forming formazan.

Devices, as used herein, refers to stationary support surfaces ofmaterials such as cellulose, plastics, collagen and polymers capable ofattachment to selenium. Examples of such devices include prostheticheart valves, intraocular lens implants, penile implants, catheters,vascular shunts, and other prosthetic implants having a specializedfunction. For example, the surface of intraocular lens implants arelinked to the selenium compound so that, upon implantation, the seleniumcatalyzes locally the production of superoxides on the surface of theimplant. The superoxide reacts with the cell surface to minimize scarformation. A vascular implant, for example, may be coated with theselenium compound so that, upon implantation coagulation of blood at theimplant site is inhibited. The surface of these implants provides amatrix for binding of the selenium compound and site specific deliveryof selenium. The effect is localized. Further, any selenium that mightescape is in amounts too small to be toxic, and is easily metabolized.Superoxide has a very short half life. It will degrade relatively soon,so it will only do considerable damage at the localized site of itsgeneration.

The non-metal element selenium exists in several catalytic andnon-catalytic oxidation states, in vitro and in vivo. If present insufficient concentrations of thiol compounds, selenium compounds such asselenides, RSe⁻, oxidize thiols, producing superoxide (O₂.⁻) and otherbiologically reactive oxygen species. Superoxide and the other producedreactive products, hydrogen peroxide, thiol radicals and other organicfree radicals are toxic to biological membranes, molecules and cells.When present in sufficient concentration as the selenoselenide anion,RSe⁻, selenium can arrest and kill normal cells, cancer cells, bacterialcells, yeast cells and viruses. When organic selenium compounds arecovalently attached to any targeting molecule such as a mono- orpolyclonal antibody, peptide or polypeptide, hormone, vitamin, drug, ordevice, such conjugates comprise a new class of pharmaceuticals anddevices that produce free radicals. Selenium is uniquely different fromother elements that produce free radicals, i.e., iron, copper or cobalt,in that selenium can readily form small adducts replacing sulfur and itcovalently combines with carbon and hydrogen compounds. Such seleniumlabeled adducts of the proper chemistry will remain non-toxic untilactivated by a thiol and the free radical pharmacology can bemolecularly localized by the carrier molecule. This free radicalchemistry is also useful for competitive protein binding assays. Thefree radical chemistry generated by selenium compounds can be detectedby chemiluminescence or reduction of dyes by a spectrophotometerproviding for quantitation of a compound which binds the antibody,hapten or drug to which selenium is attached and to which itsubsequently reacts with thiols.

A series of tests were conducted to covalently attach the seleniumcompounds to a variety of carriers. Further tests were done to test theability of the selenium-carrier conjugates to produce superoxides andkill cells, or inhibit cell growth. The temperature range for attachmentof selenium compounds to carriers depends on the carrier used, but ingeneral will be between 4° C. to about 37° C., and preferably betweenabout room temperature and 37° C.

EXAMPLE 1

Synthesis of a Cytotoxic Selenium Carrier Antibody

A human erythrocyte antibody (available from Dako corporation)containing 5 mg protein/ml was incubated with a 1500 fold molar excessof Na₂ IO, in potassium-phosphate buffer at pH 6.0 for 1-2 hrs at aboutroom temperature. To this solution was added 100 μg of glycerol for theremoval of excess periodate and, following dialysis, a 200 fold molarexcess of selenocystamine-HCl (available from Sigma Chemical Co.)containing a 1:2.2 molar ratio of triethylamine. This method in itsgeneralized procedure is that described by Hurwitz, E., Levey, R.,Mason, R., Wilcheck, M., Aron, R. and Sela, M., "The covalent binding ofdaunomycin and adrionmycin to antibodies with retention of both drug andantibody", Cancer Research, vol. 35, pp. 1175-1181 (1975) and Hurwitz,E., "Specific and nonspecific macromolecular-drug conjugates for theimprovement of cancer chemotherapy", Biopolymers, vol. 22, pp. 557-567(1983), by O'Shannessy, D. J. and Quarles, R. H., "Labeling of theoligosaccharide moieties of immunoglobulins", J. Immunologic Methods,vol. 99, pp. 153-161 (1987) and by Rodwell et al., U.S. Pat. No.4,571,958, issued Jun. 9, 1987.

In this reaction the selenocystamine of the configuration (RSeSeR) formsa stable covalent shift base and covalent bond between the selenoamineand the periodate-oxidized carbohydrate aldehyde moiety upon reductionwith cyanoborohydrate. The antibody-selenium conjugate (Ab-Se) was thenseparated from the unreacted components upon a column of Shephadex G-25which was eluted and detected by a UV monitor at 280 nm. Followingextensive dialysis, the Ab-Se continuously produced superoxide (O₂.⁻) atpH 7.2 or pH 9.0 as monitored by lucigenin (available from SigmaChemical Co.) chemiluminescence, a well known technique, in the presenceof glutathione using a Los Alamos Diagnostics Luminometer.Chemiluminescence, which is specific for O₂.⁻, can be entirely quenchedin this reaction by native superoxide dismutase.

EXAMPLE 2

Cytotoxicity of the Ab-Se to Human Erythrocytes

Aliquots of the Ab-Se at various concentrations, i.e., in a doseresponsive manner, were incubated at 37° C. for 14 hrs. with a 1%suspension of washed human erythrocytes in a physiologically balancedsolution for maintenance of cell viability. At this time erythrocytehemolysis is measured at 415 nm by absorption of released hemoglobin asdescribed by Hu, M. L. and Spallholz, J. E., "In vitro hemolysis of raterythrocytes by selenium compounds," Biochemical Pharmacology, vol. 32,pp. 957-961 (1983).

EXAMPLE 3

Comparative Examples of Cytotoxicity to Erythrocyte Membrane

In a second measure of cytotoxicity of the Ab-Se conjugate, thefollowing conditions were performed to visually assess cytotoxic damageto the erythrocyte membrane. A 1% suspension of human erythrocytes wasincubated at 37° C. for 3 hours with high levels of the Ab-Se carrierprepared as in Example 1. Then the cells were examined for membranedamage using an Olympus Microscope BH with the following optics; 4×,10×, 40× and 100×.

The experiment consisted of samples of:

1) control erythrocyte cells with no antibody;

2) erythrocyte cells with Ab-Se;

3) erythrocyte cells with native Ab;

4) erythrocyte cells pre-incubated with native Ab for twenty minutesfollowed by the addition of Ab-Se at 37° C.; and

5) erythrocytes incubated for 3 hrs. at 37° C. with selenocystamine atthe same selenium concentration as the Ab-Se, i.e. 0.5 μg Se per Ab-Seconjugate.

The following are descriptions of the appearance of the erythrocytecells following 3 hours of incubation as followed optically.

1) The control cells all appeared of normal size and shape; they had atypical erythrocyte biconcave disk appearance with a smooth membranethroughout.

2) The erythrocyte cells with Ab-Se revealed extensive lysis by theappearance of cells, with extensive swelling, up to 4-6 times controlcell size; the cells also revealed prominent multiple or single membraneholes with extruded cytoplasm; the cells that remained intact revealedextensive burring throughout the membrane with full spike-like edges inappearance, evidence of lipid peroxidation damage to membranes from freeradical activity.

3) The erythrocyte cells with the native Ab all appeared of normal sizewith perfectly smooth cell membranes and no evidence of visual membranedamage; the cells tended to clump together in large arrays of cellmasses. This effect is due to the native Ab forming links between cells,i.e., cell-Ab-cell-Ab-cell-Ab, etc. aggregates. There was no lysis ofcells.

4) The erythrocyte cells preincubated with native Ab for twenty minutesprior to the addition of Ab-Se appeared identical to #3 above, revealingno cellular membrane damage.

5) The erythrocyte cells incubated with selenocystamine.HCL in equal Seconcentrations to Ab-Se showed no lysis of cells yet there appeared tobe a small percentage of cells, ca, 5%, with burrs revealing evidence ofonly minor toxicity to membranes.

This example demonstrates that the Ab alone causes no damage to thecells and that the selenium compound alone, without conjugation to thecarrier, causes little or no damage to the cells. When attached to thecarrier, as in the Ab-Se, however, the combination causes extensive celldamage. The example in which the cells were preincubated with Ab-Se mustspecifically bind to the target site to react with thiols and generatesuperoxide. The preincubation step allows the native Ab to block thebinding sites. Low levels of native Ab will inhibit the Ab-Se binding.Higher levels of native Ab will stop Ab-Se binding completely.

FIG. 1 shows that for the same series of comparative tests as those ofExample 2, high levels of Ab-Se produced nearly 100% hemolysis at 14hours, 85% occurring within the first four hours; whereas in theAb/Ab-Se example having a ratio of 1:1 native Ab to Ab-Se an equalamount of unlabeled native antibody blocked the binding site of theAb-Se causing a 70% reduction in hemolysis. When the ratio of native Abto Ab-Se was increased to 2.5:1, the percent hemolysis decreaseddramatically. Control cells in PBS and in the presence of nativeantibody alone exhibited only ca. 8% and 6% spontaneous hemolysis,respectively.

EXAMPLE 4

Synthesis of a Selenium Containing Cytotoxic Cancer Antibody B72.3

An immunoglobulin specific for a widely expressed cancer antigen,TAG-72, originally obtained from colorectal cancer cells, but found in awide variety of other cancers, has been modified by the covalentattachment of selenocystamine and shown to produce superoxide in thepresence of thiol in vitro and bind and be cytotoxic to cancer cells invitro. In general, 5 mg/ml B72.3 antibody specific to the TAG-72antigen, produced by well known means in a mouse, is oxidized with 0.42mg of NaIO₄ for 1 hour at 0° C. 100 μl of glycerol is then added. Theoxidized B72.3 antibody is reacted with a 200 fold excess ofselenocystamine at 4° C. for 48 hours. The antibody-Se complex is thenreduced by 100 fold excess of cyanoborohydride and then chromatographedon a G-25 Sephadex column by known techniques. The antibody monitored at280 nm is collected, dialyzed and concentrated on a Protein G column.The antibody-Se immunoconjugate is shown to produce superoxide (O₂.⁻) invitro using amplified lucigenin-chemiluminescence. The chemiluminescencecan be inhibited by addition of superoxide dismutase.

When this cancer antibody is added to cancer cells grown in tissuecultures in 24 well plates, the antibody-Se immunocomplex is shown to becytotoxic to the cancer cells grown in cultures done in a dose dependentmanner over two days and shown in FIG. 2 as measured by cellularincorporation of tritiated thymidine.

EXAMPLE 5

Cytotoxicity of Selenofolate

Another example of this technology is the synthesis of selenofolate fromthe vitamin, folic acid and selenocystamine.HCl of the configuration,Folate-SeSeR. Into a round bottom flask with stoppers and a stir bar isadded 26 mg of selenocystamine.HCl, 44 mg of folic acid and 26 μl oftriethylamine in dry (20 ml) chloroform. To this suspension was added 23mg of dicyclohexylcarbodiimide. The mixture was magnetically stirred for48 hours after which 4 ml of water was added. The reaction mixture wastransferred to a 250 ml round bottom flask and dried using a Buchii 1rotoevaporator. The dry yellow-orange content was washed with distilledwater, which was discarded, and then redissolved in warm methanol. Thecontents in three aliquots was chromatographed on a 28.0×2.4 cm columnof Sephadex LH-20 in methanol. The product, selenofolate was collectedbetween elution volume 71-104 ml. Unlike folic acid, selenofolate is notfluorescent under black (UV) light. The product (very pale yellow-whitecrystals) was crystallized from hot methanol. The Rf of selenofolate inmethanol on 60 Å silica gel TLC is 0.65; the Rf of folic acid andselenocystamine is 0.00. Spectral data in 100% methanol for the product,selenofolate, and the reactants are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        SPECTRAL DATA OF SELENOFOLATE AND REACTANTS                                                          Maximum       Minimum                                    Color     Absorption  Absorption                                            ______________________________________                                         Folic Acid      Bright                                                                              286 nm    251 nm                                          Yellow           348 nm                                                      Selenocystamine Bright          302 nm        265 nm                          .HCL      Yellow                                                              Selenofolate    Very Pale        276 nm        259 nm                          Yellow                                                                     ______________________________________                                    

The product, selenofolate of the configuration Folate-N--CH₂ CH₂ SeSeCH₂CH₂ NH₂, produces superoxide (O₂.⁻) in the presence of glutathione asmeasured by lucigenin chemiluminescence. This modified vitamin compoundis cytotoxic to cells upon uptake in a dose dependant manner.Derivatives of folate are commonly used chemotherapeutic drugs, asdescribed in Stokstad, E. L. R. and Jukes, T. H. "Sulfonamides and folicacid antagonists: A historical review", J. Nutrition, vol. 117, pp.1335-1341 (1987); Kramer, K. G., Bell, R. and Pieteraz, G. A.,"Aminopterin-Monoclonal antibody conjugates: Antitumor activity andtoxicity", Drug Delivery, vol. 1, pp. 29-33 (1993).

EXAMPLE 6

Attachment of Selenocystamine: HCl to a Cellulose Matrix Device

Into a screw-topped 50 ml test tube is added 10 pieces of a cellulosepad of approximately 6 mg each and 34 mg of Na₂ IO₄ adjusted to pH 6.0in 20 ml. Periodate oxidation is carried out at room temperature for 4hrs after which the cellulose pads are drained and washed 5 times withpH 6.0 buffer. To the cellulose pads in 25 ml of PBS buffer pH 6.0 isadded 21 mg of selenocystamine.HCl and 30 μl of triethylamine containedin 5 ml of buffer. Beginning at 0 time (control), 1/2, 1, 2, 3, 4, and 8hours thereafter, a cellulose pad was removed from the buffer, washedwith distilled water and air-dried. Three pads were collected at 8 hrs.,washed and air-dried. As shown in Table 3, the capacity of the cellulosepad, cellulose-NH--CH₂ CH₂ SeSeCH₂ CH₂ NH₂, to generate superoxide(O₂.⁻) as measured by lucigenin chemiluminescence at pH 7.2 in thepresence of glutathione is generally proportional to the incubation timeof the oxidized cellulose pad with the selenocystamine.

                  TABLE 3                                                         ______________________________________                                           CHEMILUMINESCENCE OF                                                          SELENOCYSTANINE LABELED CELLULOSE PADS                                             Time of Reaction (hrs.)                                                                     Avg. CL units mg Cellulose/Min                          ______________________________________                                        0              0                                                                0.5 251                                                                       1.0  320                                                                      2.0    459                                                                    3.0 443                                                                       4.0                         427                                               8.0                           510                                           ______________________________________                                    

Additional results demonstrate that the selenium continues to work,without significant variation over time.

FIG. 5 demonstrates the ability of the selenium compounds, when bound toa cellulose sponge, to significantly inhibit the growth of the otherwisefast growing mouse fibroblast cell line, NIH-3T3. The NIH-3T3 cells weregrown in plates of calf serum, a standard mixture for stimulatinggrowth. To these plates, sponges prepared as in Example 6 were placed.The experiment was run twice. Each time, cell growth was significantlyreduced compared to the control cells not exposed to theselenium-carrier conjugate.

The highest labeled cellulose-selenium pad has been experimentallyimplanted into the eye of a New Zealand rabbit undergoing primaryfiltration surgery that mimics human refractory glaucoma. Trabeculectomyfailure due to refractory glaucoma is due to fibrosis and scarring ofthe bleb (a pocket created when tissue sewn back after the opening ismade in the anterior chamber of the eye) at the episcleral level. Inrabbits, implantation of a piece of the selenium-labeled cellulose padof ca. 1×0.5 nm under the bleb in comparison to control surgery (a shamoperation wherein a cellulose pad not labeled with selenium is implantedin the left eye vs. implantation of the selenium coated cellulose pad inthe right experimental eye) has shown the following experimentaleffects; 1) the experimental eye intravascular pressure wassignificantly reduced following surgery and took a longer time to returnto normal then the control eye, and 2) the experimental eye with theSe-cellulose pad upon pathological examination showed no fibrosis nearand within the pad as compared to the control eye with a cellulose pad(no selenium) which had fibrosis. These results show that thecellulose-selenium labeled pad prevents scarring due to fibrosis inrabbits. Scarring due to fibrosis is the main cause of surgical failurein human refractory glaucoma. The cellulose-selenium pad preventedlocalized formation of scar tissue growth in the rabbits tested withoutpathological observable damage to normal tissues.

EXAMPLE 7

Attachment of Selenium Containing Compounds to Plastic Results in aMaterial That Inhibits Cellular Growth

Plastic is treated with an ion flux of CO₂ plasma in a gas plasmamachine produced by Advanced Plasma Systems, Inc. (St. Petersburg, Fla.). This causes the CO₂ to react with the surface layer of atoms toproduce carboxyl groups on the surface of the plastic. These carboxylgroups can then be covalently crosslinked by well known techniques, suchas those used for peptide synthesis coupling amino groups to a carboxylgroup, to selenium containing compounds such as selenocystamine by theuse of 1,ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.This technique will work for any plastic material including teflon. Theplastic containing the covalently attached selenium compound is found toinhibit the growth of cells growing in tissue culture. These results canbe seen in FIGS. 3 and 4 where untreated plastic is shown to sustain thegrowth of cells while the plastic with the selenium attached is shown toinhibit cellular growth.

EXAMPLE 8

Synthesis of the CD-4 Selenopeptide

The HIV virus, which causes AIDS, is known to attach to and destroy thefunction of helper-T-cells, thereby undermining the immune system. Thepeptide which represents the segment of the CD4 protein on the surfaceof helper-T-cells to which the HIV (AIDS) virus attaches, TYICEVEDQKEE,was synthesized. This was modified for better binding to the AIDS virusresulting in the benzylated form: TYIC_(bzl) E_(bzl) VEDQKEE.Selenoproprionic acid was then activated by reaction with acarbodiimide, such asEDC[1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide]. This activeO-acylisourea intermediate was then reacted with the above peptide toform an adduct that would target the selenium for the AIDS virus.

Tests are underway to demonstrate that the peptide-Se conjugate willspecifically bind to the outer surface of the HIV virus, catalyze theproduction of superoxide upon contact with thiols on the surfaceproteins of the virus and kill the virus. As stated previously, themechanism of this invention eliminates the problems encountered due tothe lack of an uptake mechanism in viruses. It is hoped that thisprocedure will also avoid the problems associated with the HIV viruses'ability to mutate and thereby confound previous attempts at killing thevirus or treating AIDS.

EXAMPLE 9

Free Radical Diagnostic Applications

Selenium is particularly adapted and has great compatibility withvarious biochemicals as shown above for the labeling of antibody andfolate in Examples 1 and 5, respectively. It is known that free radicalsare cytotoxic. The application of this newly described chemistry forselenium conjugates, i.e., the generation of superoxide O₂.⁻ in thepresence of thiols allows for detection limits of 10⁻¹⁰ to 10⁻¹² gramsper millimeter by chemiluminescence or by reduction of a dye, such as,methylene blue. As stated above, methylene blue and cytochrome C in theoxidized form may be reduced by selenium attached to a receptormolecule, through the generation of superoxide. The amount of reducedmethylene blue or cytochrome C can be measured spectrophotometricallyand quantitated, thereby reflecting the concentration of the molecule towhich selenium is attached.

The present invention improves upon the invention set forth in U.S. Pat.No. 4,341,757 in that 1) no chemical degradation of the seleniumcontaining conjugate is required for analysis, and 2) that thechemiluminescence or dye detection method extends the analyticalsensitivity to 10⁻¹⁰ to 10⁻¹² grams per millimeter as time amplifies thelow concentration of selenium for detection owing to its catalyticnature. U.S. Pat. No. 4,341,757 disclosed classes of selenium compoundswhich have a high reactivity for proteins, polypeptides and antigens anddescribed the procedure for competitive protein binding assays using a"cold" non-isotopic immunoassay. The present invention extends thedetection limits and simplifies the chemistry for stable seleniumisotopic immunoassays.

While several theories exist why selenium is carcinostatic and cytotoxicto all cell in vitro and systemically toxic to animals in vivo, onlyrecently has Spallholz, J. E., "On the nature of selenium toxicity andcarcinostatic activity," Free Radical Biology and Medicine, vol. 17, pp.45-64, (1994) provided a comprehensive experimental and theoreticalunderstanding of selenium toxicity as a preoxidative catalytic generatorof superoxide (O₂.⁻) and other reactive oxygen descendants. Nopublication prior to Yan and Spallholz, "Generation of Reactive OxygenSpecies From the Reaction of Selenium Compounds With Thiols and MammaryTumor cells", Biochemical Pharmacology, vol. 45, pp. 429-437 (1993) hasdiscussed this free radical chemistry of the simple selenium compoundsin the presence of cancer cells.

The understanding and application of this catalytic free radicalselenium chemistry by covalent conjugates of only certain configurationsof selenium compounds, RSeH, RSeSeR, and RSeSeR' which is targeted tomembranes by conjugation, such as monoclonal and polyclonal antibodies,peptides and other molecules such as folate and devices such as acellulose pad or plastic implant, provides for a targeted seleniumtoxicity that occurs only in a macromolecular environment withoutsystemic selenium toxicity. Those skilled in the art familiar withtargeting molecules (as described in Sela, M. and Hurwitz, E.,"Conjugates of antibodies with cytotoxic drugs", Immunoconjugates,Antibody Conjugates in Radioimaging and Therapy of Cancer, pp. 189-216(C-W Vogel, ed.)(Oxford University Press (1987); Dillman et al.,"Comparisons of Drug and toxin immunoconjugates", Antibody,Immunoconjugates and Radiopharmaceuticals, vol. 1, pp. 65-77 (1988);and, Koppel, G. A., "Recent advances with monoclonal antibody drugtargeting for the treatment of human cancer", Bioconjugate Chemistry,vol. 1, pp. 13-23 (1990)), such as molecular conjugates of antibodies,peptides, vitamins, liposomes or other targeting agents, will appreciatethe pharmacological importance of this targeting selenium free radicaltechnology.

This invention employs the full understanding of the free radicalchemistry of specific selenium compounds. While these compounds aresystemically toxic this invention utilizes this toxicity in a controlledmanner by attaching the selenium compound to a targeting molecule suchas an antibody, a binding protein or peptide, or a matrix such ascellulose or plastic. This allows for the selective destruction orinhibition of cellular growth without systemic toxicity. For example wefind that the selenium-antibody immunoconjugate is only toxic when it isbound to the cell. This was shown by the fact that when its binding siteis occupied by the native antibody no selenium toxicity resulted. Wealso find that this selenium-antibody conjugate can cause its toxicityfrom the outside of the cell. In contrast, the non-covalently attachedselenium must enter cells to cause toxicity and it can do this to anycancer or normal cell. This insight into the mechanism of seleniumtoxicity (a selenium-conjugate bound to the outside of a cell is toxicand a selenium-conjugate not able to attach to a cell is not toxic) isnovel and allows us to design selenium conjugates that are also toxic toviruses.

What is claimed is:
 1. A pharmaceutical agent for the treatment ofcancer tumors and pathogenic infections comprising:a selenium-carrierconjugate for localized delivery and attachment to a target sitecomprising cancer tumors and pathogens having endogenous thiolcompounds, wherein said selenium-carrier conjugate comprises (i) anorganic selenium compound selected from the group consisting or RseH,RseR, RseR', RseSeR and RseSeR', wherein R and R' are the same ordifferent and each is an aliphatic residue containing at least onereactive group selected from the group consisting of aldehyde, amino,alcoholic, phosphate, sulfate, halogen or phenolic reactive groups andcombinations thereof, covalently attached to (ii) a carrier having aconstituent which forms a covalent bond with said reactive groups ofsaid selenium compound to produce said selenium-carrier conjugate, saidcarrier attaches to said target site for the localized generation ofsuperoxide for localized destruction of pathogens or tumors at saidtarget site; wherein said carrier comprises monoclonal antibodies,polyclonal antibodies, polypeptides, peptides, carbohydrates, lipids,vitamins, drugs, lectin, plasmid, liposome, nucleic acids andimplantable devices.
 2. The agent recited in claim 1 wherein saidpathogens comprise viruses, bacteria, protozoa, rickettsia, yeast,mycoplasma and fungi.
 3. The agent recited in claim 1 wherein saidcarrier is a F(Ab⁻)2 fragment of an antibody specific to the targetsite.